Currently this project focuses on five key areas: (1) the chemical synthesis of analogs of nitroimidazoles such as PA-824, (2) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mtb, (3) the synthesis and evaluation of inhibitors of synthesis of the mycobacterial siderophore, Mycobactin, (4) the synthesis and evaluation of substrates for, and inhibitors of, transpeptidases that are responsible for remodeling the TB cell envelope, and (5) the synthesis of inhibitors of NAD synthetase. In Project (1) we are synthesizing analogs of nitroimiadazooxazines and nitroimidazooxazoles related to PA-824. PA-824 is currently in Phase II studies in humans for the treatment of tuberculosis. Working with scientists at the Novartis Institute for Tropical Diseases, TBRS scientists have synthesized and characterized a large collection of analogs of these compounds and are advancing these through preclinical studies. Two candidate molecules were identified this year using computational methods (QSAR modeling) and were synthesized and found to be extremely potent (fourty fold better than PA-824) with improvements noted in solubility and toxicological profiling. These are currently in advanced preclinical testing and may advance to IND-enabling preclinical studies during 2010. In addition the enzyme responsible for activating these drugs has been conclusively identified and the mechanism of the drug class has been established. In this work we established the mechanism of action of PA-824 by synthesis of analogs and metabolites and in the process discovered an entire new family of enzymes and an entirely novel and unexpected chemical mechanism of drug action. These agents function as "Trojan horses" and intracellularly release Nitric oxide (NO) after activation by a bacterial enzyme. NO is a molecule intimately involved in natural human defense against tuberculosis and many other pathogens and this project suggests a new general paradigm for antibacterials development, as a result this work was published in Science. There are also immediate practical applications, in collaboration with scientists at the Genomics Institute of the Novartis Foundation (GNF) who have reengineered this protein into a soluble form and now have crystal structures of the apo enzyme as well as the enzyme with various substrates and cofactors. In Project (2) we are expanding on hit molecules obtained from quantitative high-throughput screening (qHTS) done in collaboration with NCGC. Chemi-informatic analysis of hit molecules from a titration screen has been used to cluster molecules and obtain related series of molecules. This analysis is followed by re-synthesis of hits and closely related structures for use in target identification and to explore the structure-activity relationship of molecules within a hit cluster. Five clusters of highly promising hits are being pursued by parallel library synthesis coupled to biological assay validation. In Project (3) we are evaluating approaches to the inhibition of the biosynthesis of the iron-acquiring siderophore of Mycobacterium tuberculosis, Mycobactin. In collaboration with scientists at the Unvieristy of Minnesota's Center for Drug Design we are testing inhibitors of one of the earliest biosynthetic steps in this biosynthetic pathway. Targetting iron acquisition builds upon a strong historical interest in TBRS in understanding the biosynthesis of this molecule which is required for bacterial growth during infection. we have designed and synthesized many analogs and several are being tested in animal models presently. In Project (4) we are exploring the biological role of a unique family of L,D-transpeptidases that are thought to contribute to the inherent beta-lactam resistance of TB. By a combination of biochemical and chemical studies we have established the function of two of the five members of this family and we have synthesized a panel of inhibitors and substrates in the hopes of both elucidating the underlying biology/enzymology as well as developing proof of concept molecules for a TB-specific family of penicillin-like compounds. Working with scientists at the Albert Einstein College of medicine who have solved the x-ray Crystal structure of the TB Beta lactamase in complex with clavulanic acid we have now also initiated a structure-guided program for designing better inhibitors. In project (5) we are working with the Gerratana laboratory who have solved the x-ray crystal structure of NadE which performs the last step of Nicotinamide biosynthesis to design and synthesize inhibitors of this enzyme. Both computation and structure-based approaches are being applied to elaborate fragments bound at the enzyme active site. In addition a high-throughput screening assay for this enzyme has been designed and implemented together with the NIH Chemical Genomics Center and will be run this year.